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Keywords:

  • paranodal loops;
  • myelin balloon formation;
  • splitting of myelin sheath;
  • 21.5-kDa MBP

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Recent studies have revealed a significant decrease in white matter volume, including loss of myelin, with age but minimal decrease in gray matter volume (Guttmann et al., [1998] Neurology 50:972–978). Myelin is necessary for the rapid conduction of impulses along axons. Myelinated nerve includes various domains, the node of Ranvier, the paranodal region, the juxtaparanodal region and the internode. The paranodal junction may serve to anchor the myelin sheath to the axon. We analyzed the ultrastructure of the paranodal region in myelinated fibers from the aged rat brain. Severe alterations of myelinated fibers were observed in 31-month-old rats, resulting in the appearance of macrophages, splitting of the myelin sheath, myelin balloon formation and separation from the axon. Many paranodal retractions of myelinated axons occurred in the aged rats. It should be noted that the paranodal junction is functionally important, serving to anchor the myelin to the axon and that there is a diffusion barrier in the paranodal region. We analyzed myelin-related proteins from young and aged rat brains. The 21.5-kDa isoform of myelin basic protein (MBP) almost disappeared in the 31-month-old rats, whereas other myelin proteins were not significantly changed between young and aged rats. These results suggest that this isoform, a highly cationic charged major dense component protein that binds lipid bilayer in the membrane, may participate in the formation of a paranodal diffusion barrier at the myelin/noncompact membrane border. © 2002 Wiley-Liss, Inc.

The rodent nervous system undergoes a number of morphological changes with aging including atrophy and loss of neurons and hypertrophy of glia and myelination. Recent studies have demonstrated that during aging, the cortical gray matter is preserved, whereas a significant loss of the white matter occurs; in particular, myelin breakdown occurs in the white matter of normal and healthy adults (Wickelgren, 1996). The formation of the myelin sheath results from highly specialized interactions between axons and myelinating glial cells, which are oligodendrocytes in the central nervous system (CNS) or Schwann cells in the peripheral nervous system (PNS) (Morell et al., 1999). Myelin is necessary for the rapid conduction of impulses along axons. The loss of myelin might contribute to cognitive deficits of aging, which are possibly due to the decline in the nerve cell function upon myelin atrophy (Albert, 1993). It follows that perturbation of myelin-forming glial cell function is likely to have severe consequences of neurological diseases. It is unclear, however, whether age-related alteration of the nervous system is due to a unique aging process or to the summation of the effects of known degenerative diseases. Myelinated axons contain regularly spaced unmyelinated gaps known as nodes of Ranvier. Each node of Ranvier is flanked by paranodal regions, where the axolemma is in close interaction with the membrane of the terminal loops of myelinating glial cells, also called paranodal loops. The axolemma and the membrane of glial terminal loops are tightly apposed and joined by a ridge-like sheath around the axon. Under electron microscopy, these appear as transverse bands, comprising rows of regularly spaced particles in glial cells and axonal membranes (Rosenbluth, 1995). In particular, the concept of the paranodal region has raised intriguing questions regarding the importance of axo-glial junctions with respect to nerve physiology that occurs during aging in the rat. At the paranode, myelin loops terminate and engage in the formation of a septate-like adhesion junction with the axon membrane (Peles and Salzer, 2000). This also acts as an electrical and a biochemical barrier between nodal and internodal membrane compartments (Chiu and Ritchie, 1980, Funch and Faber, 1984). The disruption of axo-glial contacts in myelinated nerve results in severe pathological conditions that have been characterized in various mouse mutants (Rosenbluth, 1980, 1990, Wang et al., 1995). Thus, elucidation of the characteristics of the axo-glial junction will contribute significantly to the understanding of myelin dysfunction in old age. We describe an age-related alteration of the paranodal junction in myelinated fibers and the molecular nature of myelin sheaths.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Animals and Tissue Samples

The 31-month-old and 1-month-old male Fisher 334 rats were supplied by the Department of Animal Science of our institute. All experimental protocols were approved by the Tokyo Metropolitan Institute of Gerontology Animal Care and Use Committee according to the protocol of the Institute of the Health Animal Care and Use.

Antibodies

The anti-myelin basic protein (MBP) monoclonal antibody used was from Boehringer Mannheim (Germany), the anti-Fyn monoclonal antibody from Transduction Laboratories (Lexington, KY), the anti-myelin proteolipid protein (PLP) monoclonal antibody from Oncogene (Cambridge, MA), the anti-CNP monoclonal antibody from Promega (Madison WI), and the anti-N cadherin polyclonal antibody from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The anti-MAG polyclonal antibody was kindly provided by Dr. Y. Matsuda (National Center of Neurology and Psychiatry, Tokyo), and the anti-F3 polyclonal antibody was a generous gift from Dr. K. Watanabe of TMIG. Anti-L1 polyclonal antibodies were used as described previously (Takeda et al., 1996).

Electron Microscopy

For electron microscopy, tissue samples from the rat cerebral hemispheres were fixed in a glutaraldehyde (1%) solution for 24 hr and postfixed in 1% osmium tetroxide for 4 hr. These tissue samples were embedded in epoxy resin. Semi-thin sections were stained with toluidine blue. Ultrathin sections stained with uranyl acetate and lead citrate were observed using a transmission electron microscope at 80 kV (JEM-100CXII, JEOL Ltd., Tokyo, Japan). In particular, the transverse or longitudinal sections (or both) of the consecutive sections of the corpus callosum portion were examined.

Histochemistry

The 1-month-old and 31-month-old rats were killed under deep anesthesia by intraperitoneal injection of sodium pentobarbital. The rats were perfused via the left ventricle with lactate Ringer's solution and phosphate buffer solution (PBS, 100 ml/100 g body weight) containing paraformaldehyde (4%). After perfusion, nerve tissues were postfixed in the same fixative for over 24 hr. Consecutive portions (coronal or sagittal section) of the rat cerebral hemispheres were examined. The samples were processed for paraffin embedding, sectioned at 7–10 μm thickness, and stained with hematoxylin and eosin (HE) or with Luxol fast blue (LFB) in preparation for electron microscopy.

Preparation of Myelin

Myelin was prepared according to the methods of Norton and Poduslo (1973). The rats were decapitated and the cerebrum was removed and weighed. Brain tissues (0.2 g) were homogenized with a Teflon homogenizer in 20 vol (w/v) of 0.32 M sucrose (4 ml), using 20 strokes. The homogenate was layered over 0.85 M sucrose in a tube (at the volume ratio of homogenate:0.85 M sucrose = 33:25), and the tube was centrifuged at 25,000 rpm for 30 min. The layer of crude myelin that formed at the interface of the two sucrose solutions was collected. The crude myelin layers were suspended in 7.2 ml of water and homogenate was centrifuged at 25,000 rpm for 15 min. The supernatant was discarded, the crude myelin pellets were resuspended in 7.2 ml of water and the suspension centrifuged at 10,000 rpm for 10 min; this washing step was then repeated twice. The myelin pellets were suspended in 4 ml of 0.32 M sucrose. This suspension was layered over 0.85 M sucrose in a tube and centrifuged exactly as described above. The purified myelin was removed from the interface with a Pasteur pipette. Statistics within figures were compared by Student's t-test and values of P < 0.05 were considered significant.

Gel Electrophoresis and Western Blot Analysis

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on 10% (for 15% MBP) polyacrylamide gels according to Laemmli (1970). To elucidate the specificity of the myelin fraction, the myelin protein concentrations were determined by the BSA assay (Pierce). A 50-μg aliquot of each myelin fraction was separated by SDS-PAGE and the fractionated proteins were stained with a silver staining II kit (Wako, Tokyo). For immunoblotting analysis, the proteins fractionated by SDS-PAGE were transferred onto an Immobilon TM PVDF membrane (Millipore, Bedford, MA). After blocking with 5% skim milk (Difco Laboratories, Detroit, MI), in Tris-buffered saline (TBS, pH 7.6) for 1 hr at room temperature (RT), the blots were incubated with several monoclonal (PLP, CNP, NCAD, Fyn, and MBP) or polyclonal (L1, MAG, and F3) antibodies as a primary antibody for 2 hr at RT. The blots were rinsed three times for 10 min with 1% skim milk in TBS, and were incubated with alkaline phosphatase-conjugated goat anti-mouse or anti-rabbit IgG (whole molecule, 1:800) for 2 hr at RT and developed with nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) solution (both from Sigma, St. Louis, MO).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Glial cells were once regarded as mere supporting tissue for neurons, but numerous studies have demonstrated that glial cells are crucially involved in the development and effective functioning of the nervous system throughout the end of life. Therefore, it follows that perturbation of the glial cell function is likely to have severe consequences. Morphological changes of myelinated fibers were observed to be most severe in the aged brain. These changes consisted of myelin balloon formation, myelin splitting, myelin infolding, formation of large dense inclusion bodies and thin myelinated fibers, reduplication and remyelination. The corpus callosum was particularly affected (histochemical data not shown). A significant change of myelin balloon formation and vacuolization of myelin lamellae in the 31-month-old rats was observed. In longitudinal sections teased fibers and the myelin balloons formed mainly along myelinated fibers. Some myelin balloons exceeded 5 μm in length in the 31-month-old-rat corpus callosum (Fig. 1). Many myelinated fibers of the 31-month-old rats had excessively thin myelin sheath given the size of their axons, and these myelinated fibers had probably been remyelinated after demyelination. Axons of remyelinated fibers often seemed smaller than compared to those of myelinated fibers with normal myelin thickness (Fig. 1). The accumulation of myelin debris and macrophages in the aged rats occurred mainly between 24–31 months of age (Fig. 2). Myelin debris was observed inside the degenerating fibers. The macrophage cytoplasm was rich in organelles containing myelin fragments (Fig. 2). We further investigated the close association between the glial and axonal membranes in the paranodal region in the aged rats, because the paranodal region seemed to be an early sign of alteration in several types of neuropathies and many neurological diseases (Maxwell et al., 1991; Griffin et al., 1996). Paranodal junction formation is functionally, serving to anchor the myelin to the axon as well as isolating the periaxonal space from the surrounding extracellular space, and possibly restricting the lateral diffusion of unevenly distributed axonal proteins (Rosenbluth, 1995). Longitudinal sections showed the frequent segmental de- and remyelination, paranodal retraction of the myelin sheath, and an increase in the number of in-folded myelin loops forming fingerlike projections into the axon in the 31-month-old rats (Fig. 3). At the paranode, myelin loops terminate and engage in the formation of a septate-like adhesive junction with the axon membrane (Peles and Salzer, 2000). This adhesive structure has multiple functions. Typically, formation of a space between myelin lamellae that split at the intraperiod line was also often observed in the 31-month-old rats (Fig. 3). Figure 4 shows the ultrathin section of the paranodal region. Compact myelin lamellae split to form teardrop shaped terminal loops that extended toward the axon. Some of the terminal loops from junctions with the axolemma were marked by periodic densities, the “transverse bands.” Some of the loops did not reach the axolemma and displayed no junctional separation between myelinated fibers of the aged rats in comparison to those of the young rats. In addition, the gap and tight junction formed by myelin-forming oligodendrocytes, which formed in the lateral edges of the sheath, were disordered in the aged-rat myelinated fibers (Fig. 4B). In general, compact myelin lamellae form tight junctions between successive terminal loops in the paranodal regions in 1-month-old rats (Fig. 5). To evaluate the molecular composition of aged-rat myelin, including the paranodal membrane specialization, we then carried out myelin constituent protein and Western blot analyses of purified myelin extracts. As shown in Figure 6, the pattern of major myelin proteins constituents on the SDS-PAGE were not changed in both 1- and 31-month-old rats. When analyzed by Western blotting (Fig. 7), most of the major myelin proteins (PLP, MAG, CNP), including some cell adhesion molecules (L1, F3, NCAD) that are known to exist in myelin, were expressed in myelin extracts prepared from 1- and 31-month-old rats, except for MBPs. The expression of MBP was weaker in the 31-month-old rats than in the 1-month-old rats. We further examined the regulation of the expression of the molecular species of MBP using our newly developed MBP antibodies, which recognized four major isoforms of MBP with molecular masses of 14.0-kDa, 17.0-kDa, 18.5-kDa, and 21.5-kDa, (Akiyama et al., 2002) (Fig. 7). These four major molecular species of MBP were observed in myelin extracts prepared from the 1-month-old rats; however, not all isoforms were expressed in the same manner as in the case of the 31-month-old rats; only the 21.5-kDa isoform of MBP was not observed in the aged animals. Although Fyn tyrosine kinase has been shown to be important for CNS myelination (Umemori et al., 1994), however, the expression of Fyn is no significant difference between the 1- and 31-month-old rats (Fig. 7). These results suggest that MBP (21.5-kDa) may be a prerequisite for the formation of a normal diffusion barrier in the paranodal region of the 31-month-old rats.

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Figure 1. a: Myelin balloons in 31-month-old-rat corpus callosum. Longitudinal sections reveal the continuity of the inner and outer portions of the myelin sheath. Ballooning (asterisks) is most common in the aged rat brain; remyelination fibers (small arrows) are also present in the aged rat. b: A higher magnification of the boxed area of a is shown in b. The myelin balloon (asterisks) exceeded 5 μm in diameter. MF, axon microfilament. Scale bars = 2 μm in a; 500 nm in b.

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Figure 2. Macrophages in 31-month-old-rat corpus callosum. Note that the cytoplasm of macrophage contains large dense bodies (large arrows), debris of myelin (arrowheads) and abundant pleomorphic lysosomes containing myelin fragments (small arrows). Scale bar = 1 μm.

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Figure 3. a: Myelin and paranodal morphology in the 31-month-old-rat corpus callosum. Electron micrographs of longitudinal sections through the paranodal region of the aged rat show that oligodendrocyte forms myelin sheath (M) around axons of 2 μm diameter. A transversely cut axon (arrowhead) is also shown. There is enhanced splitting of the myelin sheath, and many paranodal junctions, remyelinated fibers (large arrows), and myelin balloons (asterisk). Some of the terminal loops (small arrows) failed to contact the axon. b: A higher magnification of the boxed area of a is shown in b. Note that splitting of the myelin sheath (small arrows) occurs at the intraperiod line; myelin vacuolization (asterisk) is also observed. M, compact myelin lamellae. Arrowhead indicates terminal loop in the paranodal region. Scale bars = 500 nm in a; 100 nm in b.

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Figure 4. a: Ultrastructural analysis of paranodal region in myelinated axons of 31-month-old-rat corpus callosum. The axonal membrane faces the paranodal loops, but some terminal loops (one of which is indicated by a small arrow) fail to reach the axon. Many loops are unusually and widely spaced from the axolemma. NR, node of Ranvier. b: A higher magnification of the boxed area of a is shown in b. Details of the paranodal region showing compact myelin lamellae (M) split to form teardrop shaped terminal loops (one of which is indicated by a large asterisk) that extend toward the axon; the characteristic transverse bands (arrow heads) are also apparent. Some terminal loops (small arrows) fail to reach the axonal membrane and show no junction specialization. Axonal vacuole (asterisk) is also seen in the aged rat. The space for myelin splitting is also observed (small asterisk). A dotted arrow represents one myelin sheath. Scale bars = 500 nm in a; 100 nm in b.

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Figure 5. Electron micrographs of longitudinal sections of through the paranodal region in 1-month-old-rat corpus callosum. Note that compact myelin lamellae (M) split to form terminal loops that extend toward the axon. Myelin lamellae form very conspicuous tight junctions between successive terminal loops (asterisk) and transverse bands (arrowheads) in the paranodal region. NR, node of Ranvier. Diameter of axon is 1 μm. Scale bar = 100 nm.

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Figure 6. A: SDS-PAGE gel electrophoresis. Purified myelin as separated by SDS-PAGE (10% acrylamide gel) and myelin proteins were visualized by silver staining. The molecular weight markers used, from top to bottom, were: myosin (200 kDa), β-galactosidase (116 kDa), phosphorylase B (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (311 kDa), trypsin inhibitor (21 kDa), and egg white lysozyme (14 kDa). a: 31-month-old rat-myelin extracts. b: 1-month-old-rat myelin extracts. c: Molecular weight markers. B: Amount of purified myelin in young (1-month-old) and aged (31-month-old) rat brain. Note that equal amounts of cerebral hemispheres from the young and old rat were subjected to myelin purification. Myelin concentration was determined on a dry basis. Data are presented as the means ± SEM (n = 4), P < 0.05. a: 31-month-old rat myelin extracts. b: 1-month-old rat myelin extracts.

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Figure 7. A: Western blot analysis of myelin-related proteins. Purified myelin extracts from (a) the 31-month-old and (b) 1-month-old-rat brains were probed with antibodies to the proteolipid protein (PLP), myelin-associated glycoprotein (MAG), 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNP), myelin basic protein (MBP), glycosylphosphatidyl inositol (GPI)-anchored cell adhesion molecule (CAM), contactin/F3 (F3), L1 cell adhesion molecule (L1), N-cadherin (NCAD), and Src family protein tyrosine kinase Fyn (Fyn). No significant changes in the expression of the above myelin-related proteins were observed. B: Western blot analysis of purified myelin extracts from (a) 31-month-old and (b) 1-month-old, as control reference. MBP (95% purified, Upstate Biotechnology, 10 μg/lane) was applied in lane c. The asterisk indicates degraded MBP. Note that the extracts were resolved by SDS-PAGE. In each lane (a,b), 50 μg of protein was applied and the MBPs were subjected to Western blot analysis using the antibody to the MBP peptide (Akiyama et al., 2002). The four major isoforms of MBP were identified with the MBP peptide antibody, but the 21.5-kDa isoform of MBP almost disappeared in the aged rat myelin extracts (arrowhead).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Recent neuropathological studies have demonstrated that with normal aging, neuronal loss in the cortex is either not significant or not as extensive as most earlier reports suggested (Brody, 1995, Henderson et al., 1980, Anderson et al., 1983). Rather, in aged humans and nonhuman primates, a highly significant age-related decrease in volume of the white matter was observed, although the volume of gray matter showed only a relatively small percentage decrease with age (Guttmann et al., 1998). In addition, a recent study in humans based on stereological methods has revealed a significant decrease as much as 28% in the white matter volume with age but minimal decrease in gray matter volume (Pakkenberg and Gundersen, 1997). A greater loss of myelinated fibers than neurons in terms of number does not seem very likely. These recent investigations have suggested that the extent of loss of myelin around the fibers is larger than the extent of reduction in the number of nerve cells, which may partly explain the reduction in brain function with normal aging. In the present study, therefore, we attempted to elucidate how myelinated fibers degenerate with aging. Our results indicate that morphometric alteration of 31-month-old-rat myelinated fibers enhanced myelin balloon formation and myelin splitting, and increased the number of infolded loops of myelin and macrophages (Figs. 1–4). These findings are consistent with the neuropathologic reports in rats that show a loss of myelinated fibers with age (Knox et al., 1989). It is worth noting that the close association between myelin-forming glial cells and axonal membranes in the paranodal region is functionally important, serving to anchor the myelin to the axon, as well as isolating the periaxonal space from the surrounding extracellular space and possibly restricting the lateral diffusion of unevenly distributed axonal proteins (Rosenbluth et al., 1995). Furthermore, the paranodal region seems to be a region of early alteration in several types of neuropathies (Maxwell et al., 1991). These investigations may be strongly correlated with the cause and effect of myelin loss with normal aging. The findings of the present study together with those of the above studies, showing the paranodal retraction of the myelin sheath and increase in the number of infolded myelin loops forming fingerlike projections into the axon, support the notion that alterations in the white matter and myelin loss may represent the predominant neuroanatomical change in normal aging in the rat.

MBPs Are Essential for Proper Formation of Paranodal Junctions

To evaluate the molecular nature of axo-glial communication at the paranode to deepen our understanding of myelin loss in the aged rat, we carried out Western blot analysis of myelin constituent proteins prepared from purified myelin extracts. Surprisingly, only the expression of the 21.5-kDa of MBP was not observed in the 31-month-old-rat myelin sheath (Fig. 7). There were no differences in the expression patterns of myelin constituent proteins and adhesion molecules between the young and aged rat brains. Myelin in CNS contains structurally and antigenically related MBP isoforms (14.0-kDa, 17.0-kDa, 18.5-kDa, and 21.5-kDa), which are encoded by respective mRNAs (Ferra et al., 1985). The 21.5-kDa MBP has peptide sequences encoded by all seven exons. The splicing of exons 2 or 6 yields the mRNAs encoding the 18.5-kDa or 17.0-KDa MBP, respectively. The 14.0-kDa MBP isoform is obtained when both exons 2 and 6 are excised (Pedraza et al., 1997). It was demonstrated that the distribution of 14.0-kDa and 18.5-kDa MBPs in the transfectants was confined to the plasma membrane, and 21.5-kDa or 17.0-kDa MBP was distributed to the cytoplasm and the nucleus of transfectants. Pedraza et al. (2001) have reported recently that there is a diffusion barrier in the myelin sheath that function as a molecular barrier between compact myelin and the apices of the noncompact paranodal loops. In the CNS, this barrier is sealed by MBP, a very abundant, highly cationic charged intracellular protein that binds phosphatidylserine moieties, which are asymmetrically disposed in the cytoplasm of the compact myelin bilayer, and may be responsible for the stable myelin sheath together to form a compact structure. This diffusion barrier model of MBP in the myelin is consistent with the shiverer mutant mouse: a deletion of 5 of 7 exons comprising the gene encoding MBP makes the cells incapable of producing this oligodendroglial component that is essential for effective compact myelination. Thus, failure to form normally confined and highly concentrated MBP in the paranodal loops can result in free diffusion of transmembrane proteins into the defective myelin bilayer (Tait et al., 2000). Taken together, these data suggest that the consequence of significant age-related myelin loss must be due to the 21.5-kDa MBP, which is the site of MBP-mediated membrane fusion and which probably serves to immobilize the fused hemibilayers, thereby preventing transmembrane proteins that have cytoplasmically disposed domains from passing through the diffusion barrier. Investigations related to this issue will have implications for the treatment of age-related changes in many neurological diseases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors are grateful to Dr. Chika Seiwa for helpful suggestions and to Dr. K. Akiyama for anti-MBP antibody.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES